小鼠脑微血管内皮细胞的原代培养与纯化

目的:探讨纯度较高的小鼠脑微血管内皮细胞的分离与原代培养。方法:取8～14周龄BALB/c或C57/BL6小鼠,无菌分离脑组织,采用改进的两种方法即2次酶消化和1次密度梯度离心法及1次酶消化和1次密度梯度离心法分离小鼠脑微血管段,接种于涂布有Ⅳ型胶原的培养皿上进行原代培养,相差显微镜下观察细胞形态,并以免疫荧光法鉴定Ⅷ因子相关抗原。结果:改进的两种方法均可见内皮细胞在接种后12～16 h从微血管段周围爬出,5～7 d时细胞呈"漩涡状"生长,并可见细胞汇合,90%以上培养的细胞Ⅷ因子相关抗原表达阳性。结论:改进后的两种方法均能进行较高纯度的小鼠腑微血管内皮细胞的分离和原代培养。     

小鼠心肌微血管内皮细胞的原代培养及生物学特性

 目的：探讨一种高效、稳定的从小鼠心肌组织中分离培养心肌微血管内皮细胞的方法并观察细胞的生物学特性。方法：以多聚赖氨酸对培养皿作预包被, 采用Ⅱ型胶原酶消化、差速贴壁分离法,结合内皮细胞专用培养基,分离培养微血管内皮细胞并进行扩增。利用倒置显微镜观察细胞生长情况;台盼蓝法、绘制生长曲线和MTT法分别测定心肌微血管内皮细胞传代成活率、不同代生长及增殖特征;以DiI-ac-LDL和FITC-UEA-1荧光双标,结合CD31、vWF和CD34 免疫荧光鉴定细胞表面特异性抗原;体外血管形成实验检测心肌微血管内皮细胞的功能。结果：酶消化、差速贴壁法分离培养2 d后,细胞呈散在的、小簇状聚集性生长,4~5 d迅速生长呈单层排列,细胞为梭形、三角形或多角形,7～8 d后呈铺路石样即可传代;传代细胞经台盼蓝法检测成活率均为95%以上;第1、3代细胞生长曲线近似“S”形,MTT法显示第1、3代细胞在第2～4 d吸光度变化较明显（P<0.05）;随传代次数的增加,第5代细胞生长曲线近似平缓,吸光度无明显变化,细胞逐渐衰老; DiI-ac-LDL和FITC-UEA-1 荧光双标阳性率为(89.2±3.5)%,相关特异性抗原CD31、vWF和CD34 免疫荧光鉴定阳性率为(56.7±3.7)%、(78.5±2.6)%和(67.8±4.2)%;体外血管形成实验显示,6～12 h后出现明显的管腔结构。结论：以多聚赖氨酸作预包被,采用Ⅱ型胶原酶消化、差速贴壁法并结合内皮细胞专用培养基,可获得纯度较高的小鼠心肌微血管内皮细胞,为心脏疾病的研究提供种子细胞。     

大鼠肠黏膜微血管内皮细胞的体外培养

目的培养大鼠肠黏膜微血管内皮细胞,为内皮细胞的体外研究提供实验材料。方法利用机械吹打法和酶消化法分别分离出肠绒毛固有层,采用植块培养法培养原代内皮细胞,依次以局部消化法和差速黏附法进行纯化,细胞鉴定采用第Ⅷ因子相关抗原免疫荧光检测和硝酸银染色。结果成功分离培养出大鼠的肠黏膜微血管内皮细胞,纯化后的细胞可传到18代以上。结论为肠黏膜微血管内皮细胞的体外培养建立了一种操作简单、成本低廉的方法。     

改良贴壁法结合内皮细胞培养基培养小鼠肺微血管内皮细胞*☆

背景：肺微血管内皮细胞是研究微循环的重要内皮细胞模型之一,众多培养方法中单纯贴壁法操作相对简便,但耗时长,杂质细胞多,是批量培养细胞的最大障碍。 目的：建立优化的小鼠肺微血管内皮细胞体外培养方案,观察细胞生长状态并鉴定细胞性质。 方法：无菌状态下快速剪碎5日龄C57BL/6J小鼠的肺叶外周组织,肺组织颗粒贴壁法获得肺微血管内皮细胞,并用内皮细胞培养基培养。倒置显微镜观察培养细胞生长和行为状态,Ⅷ因子相关抗原免疫组化和免疫荧光进行细胞鉴定。 结果与结论：肺组织块培养24 h内可见梭形细胞爬出,传代后细胞生长迅速,形态规则呈鹅卵石状,纯度高达98%以上,结合Ⅷ因子相关抗原检测证实其为内皮细胞。结果可见联合运用肺组织颗粒贴壁和内皮细胞培养基可高效获得原代小鼠肺微血管内皮细胞。关键词：肺微血管内皮细胞;细胞培养;优化;鉴定;小鼠;血管组织工程 缩略语注释：PMVECs: pulmonary microvascular endothelial cells,肺微血管内皮细胞 doi:10.3969/j.issn.1673-8225.2012.15.007     

小鼠肺微血管内皮细胞的培养鉴定及其血管形成功能的研究

目的: 建立一种简单有效的小鼠肺微血管内皮细胞(PMVECs)原代培养方法,并对其体外血管形成功能进行研究。方法: 取小于1周龄的C57BL/6小鼠的肺组织边缘,采用组织贴块法培养原代PMVECs。经形态学观察、免疫细胞化学鉴定后,以每孔2×10⁴的密度接种于铺有基质胶的96孔细胞板,在倒置显微镜下观察(2 h 1次)其24 h 体外血管形成过程。结果: 镜下可见原代培养的PMVECs呈梭形或多角形,单层融合呈铺路石样。细胞抗Ⅷ因子相关抗原(ⅧF-Ag)染色阳性。免疫荧光显微镜下可见大部分细胞摄取四甲基吲哚碳花青标记的乙酰化低密度脂蛋白(DiI-Ac-LDL),胞质呈现红色荧光,细胞膜表面与异硫氰酸荧光素标记的异植物血凝素(FITC-BSI)结合,呈现黄绿色荧光。PMVECs接种4~6 h后出现明显的管腔结构,10~12 h呈蜂窝状结构,此后管腔结构逐渐减少。结论: 组织贴块法培养原代PMVECs操作简单,细胞得率和纯度高,并可在体外成功建立血管形成模型进行相关研究。     

微血管通透性调节的内皮细胞机制

炎症时血管通透性升高的防治一直是医学界的一个难题。在大面积烧伤时，微血管通透性升高导致大量液体和血浆蛋白渗出丢失，在组织中聚集，造成水肿。治疗方法只有根据烧伤的面积和深度大量补液来弥补液体的丢失。虽然这不失为一有效疗法，但往往不能从根本上防治水肿的发生，其主要原因是发生机理未完全阐明。影响微血管通透性的因素有很多，可分为物理性调节和化学性调节两种。影响大分子物质通过血管壁的主要物理因素包括微血管跨壁压和胶体渗透压。血流量通过改变微血管的压力间接地调节微血管通透性，血流速度本身可能因为其产生的切应…     

微血管内皮细胞的分离和培养

血管内皮细胞密切参与包括再生、发育、伤口愈合等一系列生理过程及炎症反应、糖尿病生物膜病变、肿瘤侵袭等病理生理反应 (Ｆｏｌｋｍａｎ，１９９２)。为了对内皮细胞的功能有更多了解 ,人们对人及动物内皮细胞的培养进行了探索。近年来 ,血管内皮细胞的培养已从大血管 (主动脉、脐静脉 )内皮细胞发展到微血管内皮细胞(ＭＥＣｓ)。ＭＥＣｓ的分离和培养在认识正常的血管生成和实体瘤血管新生、炎性细胞的迁移及血管病理生理等过程中发挥了关键作用。ＭＥＣｓ因器官功能不同而有不同的异质性 ,如其形态、抗原表达及对生长因子的反应等。在…     

大鼠肺微血管内皮细胞的培养

目的建立简单有效地获取大鼠肺微血管内皮细胞(PMVEC)的培养方法。方法从实验动物、培养基、植块方法、胎牛血清浓度等方面对植块培养法进行改进,应用W istar雄性大鼠的肺组织,进行PMVEC的原代培养并传代培养;通过倒置显微镜、扫描、透射电镜观察其形态,并进行免疫组织化学鉴定。结果体外培养的大鼠PMVEC呈梭形或多角形,形成单层后呈典型的鹅卵石样或铺路石样排列,获得的血管内皮细胞纯度较高,抗Ⅷ因子阳性反应,成功建立了PMVEC的原代培养方法。结论改进的植块培养法简便、可靠,获得的PMVEC纯度高,生长状态良好,保持了内皮细胞的结构和功能,可用于其功能特性的进一步研究。     

爆炸冲击波对肺微血管内皮细胞损伤作用的病理学观察

目的 :通过观察爆炸冲击波对肺微血管内皮细胞的损伤 ,了解伤后微循环功能变化的病理学基础。方法 :采用爆炸冲击波对兔损伤动物模型以及对培养肺微血管内皮细胞损伤体外模型 ,从光镜、电镜水平上观察内皮细胞病理学变化。结果 :光镜下的主要表现为肺泡出血、肺泡隔断裂、肺大泡形成以及肺间质水肿 ,电镜下可见肺微血管内皮细胞肿胀致使血管腔狭窄 ,内皮细胞间裂隙增宽 ,核肿胀、异染色质增多 ,以及线粒体肿胀、嵴排列混乱等病理变化。体外实验主要表现为爆炸冲击波对培养的肺微血管内皮细胞的剥脱性损伤。结论 :爆炸性冲击伤后肺微内皮细胞的损伤严重且广泛 ,是伤后肺微血管通透性变化与肺微循环功能障碍发生的重要病理基础。     

大鼠脑微血管内皮细胞的培养

目的:探索一种简便易行、可培养出高纯度大鼠脑微血管内皮细胞的方法。方法:1月龄SD大鼠,解剖得到大脑皮质,密度离心法获得较纯的脑微血管段后进行原代培养,传代采用差速消化和贴壁方法进行纯化。通过形态学观察及第Ⅷ因子相关抗原免疫荧光检测对培养细胞进行鉴定。结果:密度离心法分离出的大量微血管段呈"串珠样"结构,培养24h可见短梭形、多角形细胞,8～10天基本融合。第2代细胞经免疫荧光染色,第Ⅷ因子相关抗原呈阳性,阳性率达90%。结论:原代细胞采用低分子量葡聚糖加Percoll密度离心法、传代细胞采用差速消化和差速贴壁法纯化可成功培养高纯度的脑微血管内皮细胞。     

Transmission electron microscopy of mouse blastocysts activated and growth-arrested in vivo and in vitro

Summary Blastocysts from mice in a state of delayed implantation were examined by electron microscopy, either directly or after a culture period, to find out if activation and growth arrest in vitro were similar to the corresponding stages in vivo. It was found that activation in vitro, obtained by culturing the blastocysts in an outgrowth medium, was accompained by morphological signs similar to those during oestrogen activation in vivo, namely an increase in polyribosomes and glycogen granules. Analogously, growth arrest in vitro, obtained by culturing the blastocysts in a medium deprived of glucose, arginine and leucine, was accompanied by a morphology similar to that of blastocysts obtained during delay of implantation, namely scattered ribosomes of the monosome type, few membranes of endoplasmic reticulum and a lack of glycogen granules, all signs of a low metabolism. However, the morphological parallelism between the in vitro and the in vivo systems does not necessarily mean that the growth-controlling factors are the same in both cases.Activated blastocysts were transplanted to uteri of mice in delay of implantation to characterize the trophoblast ultrastructure four days after the transplantation. The experiments demonstrated that both in blastocysts activated for 24h in vivo and in those activated for 6 h in vitro the morphology reverted to that of inactive blastocysts, indicating that the functional inactivity is related to reversion into a morphologically inactive state rather than to uterine blocking of a morphologically active state.     

大鼠脑皮质微血管内皮细胞的分离和培养

目的： 本研究旨在探索1种有效分离、培养和获取较高纯度大鼠脑微血管内皮细胞（BMECs）的方法。方法: 自12 d SD大鼠脑分离出皮质，采用二次酶消化、BSA和Percoll非连续梯度离心获得较纯的脑微血管段后，接种于涂布有明胶的培养皿进行原代培养；相差显微镜观察细胞的形态学特性，进行血管内皮细胞特异性标志物Ⅷ因子相关抗原免疫组化检测。结果: 培养24 h即可见细胞从贴壁的脑微血管段周围爬出，细胞呈短梭形，集落呈典型的“鹅卵石样”，区域性单层生长，6-7 d内皮细胞开始融合，血管内皮细胞特异性标志物Ⅷ因子相关抗原表达阳性，纯度达92.6%。结论: 成功地自大鼠脑皮质分离并培养出纯度较高的BMECs，为进一步开展脑微血管内皮细胞的生物学特性的相关研究提供有用的方法。     

Electron microscopy of endothelial cells in culture: I. Transmission electron microscopy

Summary Endothelial cells in culture can be identified by structural and functional characteristics. To examine the ultrastructure of the undisturbed endothelial cell monolayer, methods are described for in situ fixation and embedment in the tissue culture flask. Subsequent sectioning and staining for electron microscopy are carried out without detachment of the cell layer from the flask.     

Local secretory response by the mouse uterine epithelium to the presence of a blastocyst or a blastocyst-like bead

Summary The ultrastructure of the uterine secretion appearing locally at the site of a blastocyst or a blastocyst-like bead was compared to the ultrastructure of the secretion present in a sterile horn of similarly treated mice. The secretion consisted of a homogeneous substance with small vesicles. The secretion was sparse and only slightly electron dense in a sterile horn, while it was more abundant and denser in a horn with a luminal object. Around a blastocyst, the secretion appeared more dense than it did around a bead, while the small vesicles of the secretion were more frequently occurring around a bead than around a blastocyst. It is concluded that a luminal object like a blastocyst is capable of eliciting a local secretory activity in the uterine epithelium, and it is suggested that this secretion is rich in hydrolytic enzymes.     

Fluorescent activated cell sorting to isolate canine microvascular endothelial cells from adipose tissue

Adipose tissue offers an abundant source for isolation of microvascular endothelial cells (MVECs). Several cell types result from the enzymatic digestion of adipose tissue, including MVECs, mesothelial cells and fibroblasts. Pure populations of MVECs must be isolated from the mixed cultures or fibroblasts overgrow the population. Canine subcutaneous or mesenteric fat is obtained during elective surgical procedures and digested with collagenase. Microvascular endothelial cells are separated from capillary fragments by Percoll gradient centrifugation and plated in culture. At confluence, microscopic identification of most cultured cells indicate the typical cobblestone morphology of ECs, while other spindle shaped cells resemble fibroblasts. Micro- vascular endothelial cells are identified by their uptake of acetylated-low density lipoprotein (Ac-LDL). Mesothelial cells, which closely resemble MVECs in morphology, and fibroblasts are removed from cultures of MVECs using a fluorescent activated cell sorter (FACS). Acetylated-low density lipoprotein tagged with a fluorescent probe, 1,1-dioctadecyl-3,3,3,3-tetramethyl- indocarbocyanine perchlorate (DiI), is incubated with the mixed cultures and the cells are sorted using a FACS. The pure populations of MVECs that result are tested immunocytochemically and are identified by their positive staining with antibodies against factor VIII related antigen. Mesothelial cells are identified by positive staining for cytokeratin 18 antibody. Contaminating fibroblasts show negative staining for smooth muscle alpha actin, cytokeratin 18 and factor VIII related antigen antibodies. This study examines the efficiency of the fluorescent activated cell sorter to obtain pure populations of MVECs harvested from adipose tissue.     

Blastocyst attachment and early invasion during oestradiol-induced implantation in the mouse

Summary Blastocysts were recovered from mice in experimentally delayed implantation 16 hrs after an injection of estrogen. When obtained from uterine horns with Pontamine Blue-positive sites, the blastocysts possessed a smooth embryonic pole and a rough abembryonic one as seen with SEM. In the embryonic pole, most of the trophoblast cells were flat while in the abembryonic region, the cells showed irregular cell extensions and rough imprints. TEM of Pontamine Blue-positive sites demonstrated that the embryonic pole had a rather smooth borderline to the epithelium, while the borderline at the abembryonic pole was more irregular with small parts of the uterine epithelium indenting into the trophoblast. The cytoplasm of the trophoblast contained a well developed endoplasmic reticulum, several mitochondria, large vacuoles, many multivesicular bodies and lipid granules. The difference in structure between the two poles of the blastocyst is presumably related to the differences in enzymatic activity between the poles as demonstrated by histochemistry and to the greater invasive capacity of the abembryonic trophoblast.     

三叉神经痛脱髓鞘改变的光镜和电镜观察

对１５例三叉神经痛患者术中撕脱的周围神经支进行光镜观察，２例进行电镜观察。可见神经纤维不同程度退行性变，肿胀、增租，轴突不规则或消失，髓鞘正常纹理不清，结构紊乱、变形，有的增生增厚呈乳头状或球状突向轴浆，有的松解、断裂呈多层状、空泡状、空网状改变，严重者出现脱髓鞘化。同时观察到三叉神经远心端病变较近心端严重，认为三叉神经的脱髓鞘改变是三叉神经痛的主要病理改变和主要病因，支持三叉神经痛的周围病原学说，并结合文献对三叉神经痛的病因、发病机制进行了讨论。     

格林-巴利综合征患者腓神经病变的光镜和电镜观察

目的：探讨格式－巴利综合征腓神经活检的光,电镜表现与临床诊断及预后的关系。方法;对２６例ＧＢＳ患者腓神经活检进行了光镜及电镜观察。结果;有髓神经纤维的呈不同程度的减少,退行性变及脱髓鞘,在病变早期髓鞘改变呈松解,变形,断裂及多层状,部分增厚突向轴浆向。     

两种微血管内皮细胞糖链表达的凝集素细胞化学染色特征

目的 明确两种微血管内皮细胞(MVECs)糖链表达的特点.方法 复苏冻存的大鼠空肠黏膜MVECs;取3日龄SPF仔猪肺组织,采用胶原酶消化法和差速贴壁法分离培养猪肺MVECs;采用凝集素细胞化学方法,检测刀豆凝集素(Con A)、菜豆红细胞凝集素(PHA-E)、蓖麻凝集素Ⅰ(RCA-Ⅰ)、番茄凝集素(LEL)、黑接骨木...     

缺氧预处理减轻内质网应激所致的大鼠心肌微血管内皮细胞损伤

目的:研究缺氧预处理(hypoxic preconditioning,HPC)对内质网应激(endoplasmic reticulum stress,ERS)所致的心肌微血管内皮细胞(microvascular endothelial cells,MVECs)损伤的影响。方法:以毒胡萝卜素(thapsigargin,TG)诱导大鼠心肌MVECs ERS,以乳酸脱氢酶(lactate dehydrogenase,LDH)漏出和细胞凋亡率检测细胞损伤,phalloidin-FITC荧光染色和内质网染色分别观察细胞骨架和细胞内质网形态变化,双向电泳-质谱技术检测TG作用后内皮细胞蛋白质谱表达变化,Western blotting技术检测ERS相关的分子表达。结果:TG剂量依赖性诱导MVECs LDH漏出和细胞凋亡,并出现内质网应激分子钙网蛋白(calreticulin,CRT)和葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)表达上调;HPC减轻TG诱导的MVECs损伤,并可以抑制TG诱导的ERS相关分子的表达上调。结论:HPC减轻内质网应激所致的大鼠心肌MVECs损伤。